Conventionally, antigen-specific lymphocytes have been detected by adding about 200,000 lymphocytes to each well of a 96-well plate and culturing the lymphocytes in the presence of antigen for from three days to a week (Methods of Detecting Lymphocyte Functions”, Junichi YANO, Michio FUJIWARA, eds., Chugai Igakusha (1994), “Methods of Conducting Immunity Experiments I, II”, Shunsuke ISHIDA, Susumu KONDA, Morosuke MOTO, Toshiyuki HAMAOHKA, eds., Nankodo (1995)). This method makes it possible to determine that an antigen-specific lymphocyte is present within a group of about 200,000 lymphocytes. However, it does not permit the identification of individual antigen-specific lymphocytes present within the group of lymphocytes.
By contrast, a method has been developed and put into practice in recent years in which fluorescent pigment-labeled antigen molecules are mixed with lymphocytes to cause the fluorescently labeled antigen to bind to antigen receptors on the antigen-specific lymphocytes, and the lymphocytes that have bound the fluorescently labeled antigen are detected with a flow cytometer (Altman J. D., Moss P. A., Goulder P. J., Barouch D. H., McHeyzer-Williams M. G., Bell J. I., McMichael A. J., Davis M. M. Phenotype analysis of antigen-specific T lymphocytes, Science, 274: 94-96, 1996). In this method, a single lymphocyte that has bound an antigen can be identified. Further, it is also possible to separate out a single lymphocyte that has bound an antigen.
However, the above detection method requires an expensive and complex device known as a cell sorter for separation. In addition, it presents the following problems:
(1) It is difficult to set the conditions in the device for separation; great skill is required to operate the device and separate cells;
(2) Due to high background noise, when the frequency of the antigen-specific lymphocyte is 0.1 percent or less, the antigen-specific lymphocyte cannot be detected;
(3) Cell separation efficiency is low;
(4) Time is required to separate low frequency cells; and
(5) Although the binding of antigen can be confirmed, the reaction by which the lymphocyte binds the antigen cannot be analyzed.
Another method of detecting antigen-specific lymphocytes has been developed in which antigen molecules bound to magnetic beads are mixed with lymphocytes to cause the magnetic bead-bound antigen to bind to the antigen receptors of the antigen-specific lymphocytes, and a magnet is used to separate the antigen-specific lymphocytes (Abts H., Emmerich M., Miltenyi S., Radbruch A., Tesch H., CD20 positive human B lymphocytes separated with the magnetic sorter (MACS) can be induced to proliferation and antibody secretion in vitro. Journal of Immunological Methods 125:19-28, 1989).
In this method, no complex device is required, cells are rapidly separated, and the binding of antigen can be confirmed. However, how the lymphocytes that have bound the antigen have reacted with the antigen (intracellular signal transmission, RNA synthesis, protein synthesis, or some other metabolic physiological reaction of the cell) cannot be analyzed. Further, antigen-specific lymphocytes with a frequency of 0.1 or less cannot be detected.
By contrast, the present inventors conducted various investigations into providing a method for detecting antigen-specific lymphocytes that does not require a complex device, separates cells rapidly, can confirm the binding of antigen, can detect antigen-specific lymphocytes of low frequency (0.001 percent or more), permits analysis of how the lymphocyte that has bound an antigen reacts with the antigen, and permits separation of antigen-specific lymphocytes. The present inventors also developed a method of detecting the antigen specificity of individual lymphocytes and recovering antigen-specific lymphocytes that are detected.
However, no microwell array chip was known that could separately detect the antigen specificity of individual lymphocytes and permit recovery of the antigen-specific lymphocytes that were detected.
Accordingly, the present inventors conducted extensive research into providing a microwell array chip that could be used in the above-described detection method and that could hold a single lymphocyte in a single microwell. The present inventors fabricated test microwell array chips in which microwells of roughly a size capable of containing a single lymphocyte were formed on a substrate surface, and conducted tests in which lymphocytes were stored (collected) in microwells and recovered from microwells. In this process, the present inventors determined that when recovering antigen-specific lymphocytes detected in an array chip in which multiple minute microwells had been provided, and in particular, when conducting detection and recovery in separate steps, the reliable recovery of antigen-specific lymphocytes without error from the microwells in which they had been detected required reliable determination of the positions of the microwells. That is, in array chips in which multiple microwells are simply arrayed, there is a problem in that it is difficult to readily determine the position of a specific microwell.
In the above process, the present inventors further determined that when the array chip was washed after collecting the lymphocytes in the microwells using a cell suspension in the course of storing lymphocytes in microwells, there was a problem in that a large number of cells ended up flowing out of the microwells, causing the collection efficiency and filling rate to decrease markedly.
Accordingly, the first object of the present invention is to provide a microwell array chip lending itself to use in the above-described detection method and permitting the storage of single lymphocytes in single microwells. In particular, Aspect I of the present invention has for its object to provide a microwell array chip permitting the ready determination of the positions of multiple minute microwells.
The second object of the present invention is to provide a microwell array chip in which cells such as lymphocytes that have been collected in microwells tend not to flow out of the microwells during subsequent washing.
The third object of the present invention is to provide a microwell array chip lending itself to use in the above-described detection method and capable of storing individual lymphocytes in individual microwells.
In particular, Aspect III of the present invention has for its object to provide a microwell array chip permitting the ready recovery of a single lymphocyte stored in a microwell. Aspect III of the present invention has the further object of providing a microwell array chip that is not limited to lymphocytes, but permits the storage of a single organic cell in a single microwell.